Mycoplasmas comprise a large group of wall-less gram positive prokaryotes that includes several pathogens of man and animals. Currently, molecular analysis of mycoplasma pathogenesis is severely hampered by 1) lack of tools to mobilize, transfer or mutagenize genes in these organisms; 2) limited understanding of adaptations used to process critical proteins secreted through or expressed on the surface of the single limiting mycoplasma membrane; and 3) lack of classic genetic approaches (complementation or auxotrophic mutations) to study basic processes such as metabolite import/export, due to incompatibilities of mycoplasma codon usage (UGA Trp), and to the requirement for complex growth medium. The objective of this proposal is to exploit and further define recently developed strategies that circumvent these limitations, in order to examine broadly applicable features of mycoplasma membrane protein processing, transport systems, a novel gene amplification system and horizontally-transmitted transposon-like genetic elements. The prototype Mycoplasma hyorhinis (and commensal species) will be used to address the following specific aims: 1) to define translocation, signal processing and anchorage of a highly immunogenic protein (P101) and a putative lipid-modified membrane protein (P37) associated with a high-affinity permease operon; and to analyze analogous transport operons detected in two other pathogenic mycoplasmas; 2) to define a novel eukaryotic-like system of gene amplification regulating expression of several potential metal-binding proteins that may be involved with the resistance of mycoplasmas to heavy metals; and 3) to assess the mobility of transposon-like and associated chromosomal elements to understand natural exchange of mycoplasma genetic information, and to develop a mycoplasma cloning vehicle. Highly integrated methodologies include standard techniques for gene cloning, sequencing, transcription, TnphoA or site-directed mutagenesis; and immunological and membrane biochemical analysis of gene products. New techniques involve transformation and detection of reporter genes in mycoplasmas. The focus on recombinant techniques, and their use in mycoplasmas to examine membrane protein processing and transport systems, is anticipated to enhance experimental access to and understanding of mycoplasma pathobiology.